Eukaryotic cells have highly conserved enzymatic systems for ligating ubiquitin (Ub) and related proteins such as SUMO to proteins. The modifications may lead to degradation of the targeted protein, usually by the proteasome but sometimes also by the lysosome. Ub and SUMO modifications are highly dynamic due to specialized proteases that remove them. Both modifiers have many crucial roles, including important contributions to human biology. Substrates include naturally short-lived regulators and aberrant ?protein quality control? (PQC) substrates. Many human disorders, including neurodegenerative diseases, diabetes, and different cancers, are associated with abnormalities in the Ub system. The system presents many potential targets for drug development against a range of diseases. In this application, the PI proposes to undertake studies on several newly discovered regulatory mechanisms, which either alter protein modification by Ub or react to changes in the dynamics of SUMO modification in striking ways. One focus is on the response of Saccharomyces cerevisiae cells to loss of a key SUMO protease called Ulp2. The cells very quickly generate a specific two- chromosome aneuploidy, but this is eventually resolved through in vitro evolution over hundreds of generations. The evolutionary trajectories are related but distinct, and deep sequencing technologies turn this into a powerful way to study the physiological mechanisms that allow cells to adapt and flourish even without a seemingly crucial enzyme. Another new research direction addresses the regulated proteolysis of an essential yeast protein that controls both proteasome translocation into the nucleus and PQC at the ribosome. Its degradation is tightly linked to its functional state. Subcellular movements of proteasomes and their degradation under starvation conditions will also be examined. A final, also new area addresses Ub system enzymes from endosymbiotic bacteria that infect humans and other species. One focus is on a deubiquitylating enzyme (DUB) from Orientia, the causative agent of scrub typhus, a highly lethal disease. Surprisingly, the DUB protein not only cleaves Ub but also binds tightly to it as well as to clathrin adaptor proteins, small GTPases, and a specific phospholipid. The many activities in this single polypeptide are proposed to be coordinated in a way that favors pathogen infection and propagation. Another DUB of great interest is from Wolbachia, bacteria that infect millions of arthropod species and exploit this unusual DUB to alter host reproduction and promote their own inheritance. Wolbachia are being deployed as agents for fighting disease vectors such as the mosquitoes that transmit dengue fever or malaria.